Development of multiple enzyme patterning within self assembling nanoclay gel towards an artificial Golgi Apparatus

Abstract

As the complexity of pharmaceutical increases, the ability to replicate the complex biochemical signalling which drives biological processes with protein patterning techniques become essential. Protein patterning is the ability to define the position of proteins in space and time and enables the precise control of biological systems by directing or recreating entirely biological functions such a tissue regeneration, protein glycosylation and cellular function.

Advanced fabrication techniques have been developed to create biomaterials patterned with proteins for a range of biomedical applications, yet these techniques remain limited by their simplistic patterns, scalability and reduction of protein activity through immobilisation technique.

Laponite hydrogel protein patterning is a new technique promising to address these issues through a reaction-diffusion method capable of micron-scale protein patterns which remain bioactive after immobilization. However, despite its potential, the underlying mechanisms controlling protein diffusion, adsorption, and pattern stability within the Laponite network remain incompletely understood.

Overall, this thesis explored three key avenues of protein patterning within
Laponite hydrogel, developing understanding of the mechanism which drives the reaction/diffusion process and expanding the use of this technique for applications in tissue engineering and biocatalysis.

The first chapter models the physical interactions that occur during the diffusion of proteins through Laponite hydrogel, proposing two models, one for the mechanical changes and a second for the reaction-diffusion process, which gives the characteristic sharp diffusion front. The Dual-sorption model is identified as a model which correctly predicts the diffusion behaviour of BSA within Laponite hydrogel.

The second chapter expands experimentation on the patterning behaviour of BMP-2, revealing its displacement within the Laponite hydrogel by BSA. To examine this observation, the influence of pH, fluorophore conjugation and protein charge on model proteins BSA and lysozyme. None of these variables changed ability to displace BSA or lysozyme suggesting reasoning through molecular charge alone is not sufficient to overcome the limitations of BMP-2 patterning, highlighting the need for more research into protein/protein/clay interaction.

The final chapter extended protein patterning to multiple enzymes, successfully
developing a method to pattern sequential rings active enzyme within Laponite hydrogel.

This achievement marks a significant step toward constructing an artificial Golgi apparatus, demonstrating the potential of Laponite hydrogel for
glycoengineering.

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Identifiers

ORCID for Jamie McGuire: orcid.org/0000-0002-4162-2987

ORCID for Jon Dawson: orcid.org/0000-0002-6712-0598

ORCID for Richard Oreffo: orcid.org/0000-0001-5995-6726

ORCID for Nick Evans: orcid.org/0000-0002-3255-4388

ORCID for Yanghee Kim: orcid.org/0000-0002-5312-3448

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